The aim of this study was to identify potential proteomic biomarkers for chronic active antibody-mediated rejection (CAMR) in kidney transplant recipients (KTRs). Among 385 KTRs enrolled in a cross-sectional multicenter study, 26 KTRs with biopsy-proven CAMR, 57 KTRs with long-term graft survival (LGS), and 10 rejection-free matched KTRs were included. A proteomic approach was employed to measure urinary extracellular vesicle (EV) changes in the KTRs. The urinary EVs were trypsin-digested using a gel-assisted protocol and quantified by label-free liquid chromatography with tandem mass spectrometry, using a data-dependent acquisition (DDA) mode. Western blot analysis was performed to confirm the protein levels for each candidate biomarker. Analysis of the isolated EV proteins revealed 93 and 97 proteins in the CAMR and LGS patients, respectively. Proteins that were identical in both groups were excluded and only high-significance proteins with a fold change of at least 1.5 were selected as candidate biomarkers. Six proteins (APOA1, TTR, PIGR, HPX, AZGP1, and CP) that were distinguishable between CAMR and LGS were selected. The proteins were confirmed by immunoblot analyses using independently acquired urinary EV samples. AZGP1 in particular was found to be a CAMR-specific proteomic biomarker that was distinguishable from the rejection-free control group with matching kidney function, duration of transplantation, and age. We identified and validated six proteomic biomarkers for CAMR and clarified one CAMR-specific proteomic biomarker in KTRs. Further clinical trials are needed before these rejection-specific biomarkers can be applied for the early prediction, diagnosis, and monitoring of the clinical response of KTRs to the treatment of CAMR.

这项研究的目的是确定肾脏移植受者（KTR）中慢性活性抗体介导排斥（CAMR）的潜在蛋白质组学生物标志物。在一项纳入多中心横断面研究的385个KTR中，包括26个经活检证实为CAMR的KTR，57个具有长期移植存活率（LGS）的KTR和10个无排斥反应的匹配KTR。蛋白质组学方法用于测量KTR中的尿液细胞外囊泡（EV）的变化。使用凝胶辅助方案对尿液电动车进行胰蛋白酶消化，并使用数据依赖采集（DDA）模式通过无标记液相色谱-串联质谱法对尿液电动车进行定量。进行蛋白质印迹分析以确认每种候选生物标志物的蛋白质水平。对分离出的EV蛋白的分析显示，CAMR和LGS患者分别有93和97蛋白。排除两组中相同的蛋白质，仅选择倍数变化至少为1.5的高重要性蛋白质作为候选生物标志物。选择了在CAMR和LGS之间可区分的六种蛋白质（APOA1，TTR，PIGR，HPX，AZGP1和CP）。通过使用独立获得的尿液EV样品进行的免疫印迹分析确认了蛋白质。尤其是AZGP1被发现是CAMR特异的蛋白质组生物标志物，与肾脏功能，移植持续时间和年龄相匹配的无排斥对照组有明显区别。我们鉴定并验证了CAMR的6种蛋白质组生物标志物，并阐明了KTR中1种CAMR特异的蛋白质组生物标志物。在将这些排斥特异性生物标记物用于早期预测，诊断，